EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

Ghrelin induces proliferation in human aortic endothelial cells via ERK1/2 and PI3K/Akt activation

The direct ghrelin (Ghr) involvement in cardiovascular (CV) system homeostasis has been suggested by the expression of its receptor in CV tissues and by evidence that ghrelin mediates CV activities in animals and in humans. Moreover, low Ghr plasma levels have been reported in pathological conditions characterized by high cardiovascular risk. In the present study, we investigated Ghr effect on proliferation of human aortic endothelial cell (HAEC) and involved transduction pathways. Our results indicate that ghrelin elicited proliferation in a dose-dependent manner (EC(50) about of 5nmol/L) in cultured HAEC, and that this effect was inhibited by the receptor antagonist (D-Lys3)-GHRP-6. Western blot experiments documented an activation of external receptor activated kinases (ERK1/2) and Akt in a dose-dependent fashion, as well as involvement of the cAMP pathway in ERK1/2 phosphorylation. Experiments conducted with appropriate pharmacological inhibitors to investigate Ghr-induced HAEC proliferation confirmed the involvement of ERK1/2 and I3P/Akt pathways, as well as the role of AMP cyclase/PKA pathway in ERK1/2 phosphorylation. Our results indicate that Ghr promotes HAEC proliferation, and thus may be a protective factor against vascular damage. The low ghrelin serum levels reported in insulin-resistant states may not be able to effectively counteract endothelial cell injury.     

Involvement of Nrf2 and Keap1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent peptides from soft-shelled turtle

The antioxidant response element (ARE) is a cis-acting enhancer sequence located in the region containing genes related to antioxidant and detoxification. Under oxidative stress, the induction of nuclear factor-E2-related factor 2 (Nrf2)/ARE is considered as a fundamental process involved in defending reactive oxygen species (ROS) and providing protection against toxic xenobiotics. In this study, we obtained seven antioxidant peptides from soft-shelled turtle and concluded that Glu-Asp-Tyr-Gly-Ala (EDYGA) is the most potent ARE-luciferase inducer. To gain fundamental insights into the role of EDYGA in oxidative stress, we evaluated the effects of EDYGA on the Nrf2/Keap1 system in HepG₂ cells. The results revealed that EDYGA modulated the Nrf2/ARE pathway by enhancing Nrf2 level through the stabilization of Nrf2, which was accomplished by a decrease in the level of Keap1. These actions eventually led to an increase in nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by EDYGA. Docking results proved that glutamate residues of peptide EDYGA directly bind to Arg 415 of Kelch domain receptor pocke. The results were helpful in understanding the antioxidant activity of peptides from soft-shelled turtle which have potential to be used in foods and drugs as functional ingredients.     

Gastrodin prevents homocysteine-induced human umbilical vein endothelial cells injury via PI3K/Akt/eNOS and Nrf2/ARE pathway

In this study, we investigated the protective effects of gastrodin (Gas) against homocysteine-induced human umbilical vein endothelial cell (HUVEC) injury and the role of the phosphoinositide 3-kinase (PI3K)/threonine kinase 1 (Akt)/endothelial nitric oxide synthase (eNOS) and NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathways. We stimulated cells with homocysteine (1 mmol/L, 24 hours) and tested the effects of gastrodin (200-800 μg/mL) on cell viability and the production of malondialdehyde (MDA), lactate dehydrogenase (LDH) and reactive oxygen species (ROS). Then, Nrf2 distribution in the cytoplasm and nucleus as well as the expression of enzymes downstream of Nrf2 was determined. Furthermore, we analysed the expression of bax, bcl-2 and cleaved caspase3, and assessed the involvement of the PI3K/Akt/eNOS pathway by Western blots. Finally, we tested the vasoactive effect of gastrodin in thoracic aortic rings. The results showed that gastrodin decreased MDA, LDH and ROS production and increased cell viability, NO production and relaxation of thoracic aortic rings. Moreover, the protective effects of Gas on NO production and relaxation of thoracic aortic rings were blocked by L-NAME but enhanced by Cav-1 knockdown, and MK-2206 treatment abolished the effect of Gas on the ROS. In addition, treatment with gastrodin increased Nrf2 nuclear translocation, thus enhancing the expression of downstream enzymes. Finally, gastrodin increased the expression of PI3K, p-Akt, and eNOS and decreased Cav-1 protein expression. In conclusion, our study suggested that gastrodin may protect HUVECs from homocysteine-induced injury, and the PI3K/Akt/eNOS and Nrf2/ARE pathways may be responsible for the efficacy of gastrodin.     

Down-regulation of connexin43 gap junction by serum deprivation in human endothelial cells was improved by (-)-Epigallocatechin gallate via ERK MAP kinase pathway

Intercellular communication through gap junctions (GJIC) plays an essential role in maintaining the functional integrity of vascular endothelium. Despite emerging evidence suggests that (−)-Epigallocatechin gallate (EGCG) may improve endothelial function. However, its effect on Cx43 gap junction in endothelial cells remains unexplored. Here we investigated the effect of EGCG on connexin43 (Cx43) gap junction in endothelial cells. The levels of Cx43 protein in human umbilical vein endothelial cells (HUVECs) cultured under serum-deprivation 48 h decreased about 50%, accompanied by decreased GJIC. This reduction can be reversed by treatments with EGCG. In addition, EGCG activated ERK, P38, and JNK mitogen-activated protein kinases (MAPKs), which were supposed to participate in the regulation of Cx43. A MEK inhibitor PD98059, but not SB203580 (a p38 kinase inhibitor) or SP600125 (a JNK kinase inhibitor), abolished the effects of EGCG on Cx43 expression and GJIC. Moreover, although both Akt and eNOS phosphorylation were time-dependently augmented by EGCG, neither PI3K inhibitor LY294002 nor eNOS inhibitor L-NAME blocked the effects of EGCG on Cx43 gap junctions. Thus, EGCG attenuated Cx43 down-regulation and impaired GJIC induced by serum deprivation, ERK MAPK Signal transduction pathway appears to be involved in these processes.     

All-<Emphasis Type="Italic">trans</Emphasis>-retinoic acid induces apoptosis in Leydig cells via activation of the mitochondrial death pathway and antioxidant enzyme regulation

In addition to playing a fundamental role in diverse processes, such as vision, growth and differentiation, vitamin A and its main biologically active derivative, retinoic acid (RA), are clearly involved in the regulation of testicular functions. The present study was undertaken to examine the direct effect of RA treatment on Leydig (TM-3) cells. TM-3 cells were cultured and treated with varying concentrations of RA for 24h. High doses of RA (1–20μM) induced a decrease in cell vitality and an increase in lipid peroxidation. RA treatment also induced a corresponding increase in apoptosis in the same cells in a dose-dependent manner. Apoptosis proceeded via the mitochondrial dependent pathway, as demonstrated by the release of cytochrome c, caspase-3 enzymatic activation and DNA fragmentation. Conversely, at physiological doses (0.1–500nM) RA did not increase lipid peroxidation or cell death and resulted in an increase of antioxidant enzyme activity.     

A component of green tea, (-)-epigallocatechin-3-gallate, promotes apoptosis in T24 human bladder cancer cells via modulation of the PI3K/Akt pathway and Bcl-2 family proteins

Bladder cancer is the fourth most common cancer in men and ninth most common in women. It has a protracted course of progression and is thus an ideal candidate for chemoprevention strategies and trials. This study was conducted to evaluate the chemopreventive/antiproliferative potential of (-)-epigallocatechin gallate (EGCG, the major phytochemical in green tea) against bladder cancer and its mechanism of action. Using the T24 human bladder cancer cell line, we found that EGCG treatment caused dose- and time-dependent inhibition of cellular proliferation and cell viability, and induced apoptosis. Mechanistically, EGCG inhibits phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulation of Bcl-2 family proteins, leading to enhanced apoptosis of T24 cells. These findings suggest that EGCG may be an important chemoprevention agent for the management of bladder cancer.     

The coffee diterpene kahweol induces heme oxygenase-1 via the PI3K and p38/Nrf2 pathway to protect human dopaminergic neurons from 6-hydroxydopamine-derived oxidative stress

In this study, we investigated the mechanisms of kahweol protection of neuronal cells from cell death induced by the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA). Pretreatment of SH-SY5Y cells with kahweol significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Kahweol also up-regulated heme oxygenase-1 (HO-1) expression, which conferred neuroprotection against 6-OHDA-induced oxidative injury. Moreover, kahweol induced PI3K and p38 activation, which are involved in the induction of Nrf2, HO-1 expression, and neuroprotection. These results suggest that regulation of the anti-oxidant enzyme HO-1 via the PI3K and p38/Nrf2 signaling pathways controls the intracellular levels of ROS.     

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.